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A Dutch kindred with the Hermansky-Pudlak syndrome (HPS) is described. We show for the first time evidence of a lowered platelet 5-hydroxytryptamine content in obligate heterozygotes. Platelet ATP and ADP levels and ATP/ADP ratio were normal in these patients. Platelet aggregation with ADP, collagen and adrenaline was within the normal range. In contrast to the homozygous HPS patients the heterozygotes are normally pigmented and none has diaphanous irides, nystagmus or a bleeding tendency. All homozygous HPS patients have the typical triad of oculocutaneous albinism, pigmented macrophages in the bone marrow and a bleeding disorder, based on a platelet dysfunction. The platelets showed the typical characteristics of a storage pool deficiency. Their platelet factor 3 availability was decreased and the aggregation patterns showed an absent second wave with ADP, adrenaline and absent collagen aggregation. Platelet ADP levels were strongly decreased in all homozygous HPS patients, whereas ATP was lowered only in 3 out of 6 HPS patients. The 5-hydroxytryptamine content of their platelets was very low (15-20% of normal).

Summary. Thrombopoietin (TPO) is known to sensitize platelets to other agonists at 20 ng/ml, and above 100 ng/ml it is an independent activator of aggregation and secretion. In studies with a perfusion chamber, TPO, between 0·01 ng/ml and 1 ng/ml, increased platelet adhesion to surface-coated fibrinogen, fibronectin and von Willebrand Factor (VWF) but not to a collagen-coated surface. Increased adhesion was observed at shear rates of 300/s and 800/s in perfusions with whole blood as well as in suspensions of platelets and red blood cells reconstituted in plasma. The by the cyclooxygenase inhibitor, indomethacin, and the thromboxane A2-receptor blocker, SQ30741, abolished the stimulation by TPO. The effect of TPO was mimicked by a very low concentration (10 nmol/l) of the thromboxane TxA2 analogue, U46619. Real-time studies of platelet adhesion to a VWF-coated surface at a shear of 1000/s showed that about 20% of the platelets were in a rolling phase before they became firmly attached. TPO (1 ng/ml) pretreatment reduced this number to

The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the gamma-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the gamma-chain segments gamma318-320 and gamma408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to gammaDelta408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: gammaDelta319-320, gamma318Asp-->Ala and gamma320Asp-->Ala. Furthermore, antibodies raised against the sequences gamma308-322 and gamma316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment gamma316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

SummaryWe previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-binding to collagen (1). The sequence of those peptides could be aligned with the sequence in vWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF. We mutated the 1129-1136 VWTLPDQC sequence in vWF to VATAPAAC. Expressing this mutant vWF (7.8-vWF) in a fur-BHK cell line resulted in well processed 7.8-vWF containing a normal distribution of molecular weight multimers. However, binding studies of this mutant vWF to rat tail, human and calf skin collagens type I, to human collagen types III and VI, revealed no decrease in vWF-binding to any of these collagens. Thus, although the N-and Q-peptides did inhibit the vWF-collagen interaction, the resulting alignment with the vWF sequence did not identify a collagen binding site, pointing out that alignments (although with a high percentage of identity) do not always result in identification of binding epitopes. However, suprisingly removal of the A3-domain or changing the vWF sequence at position 1129-1136 resulted in an increase of vWF-binding to human collagen type VI and to rat tail collagen type I, implying that these changes result in a different conformation of vWF with an increased binding to these collagens as a consequence.

SummaryA considerable inter-individual variation in half-life of infused factor VIII is observed among patients with hemophilia A. The factors contributing to this wide range in factor VIII half-life are not known in detail. We analysed the pharmacokinetics of infused factor VIII in 32 patients with hemophilia A, comprising 20 brothers from 10 families, 3 and 4 brothers from 2 families, and 5 patients from 5 single families, respectively. Multiple linear regression analysis was used to asses the effect of several variables on factor VIII half-life. We found that the pre-infusion von Willebrand factor antigen levels (vWF:Ag) were positively correlated with factor VIII half-life (r = 0.52, p = 0.002), i. e., each variable was associated with about 27% of the variance of the other. In fraternal pairs, familial clustering was significant for AB0 blood group (p < 0.001), but could not be detected for factor VIII half-lives or pre-infusion vWF:Ag levels. vWF:Ag level (p = 0.001) and AB0 blood group (p = 0.003) significantly determined factor VIII half-life, whereas age, length, bodyweight, the presence or absence of a factor VIII gene inversion, and Rhesus phenotype did not. Patients with blood group 0 exhibited a statistically significant shorter factor VIII half-life than patients with blood group A (15.3 versus 19.7 h, respectively) (p = 0.003). Patients with blood group A and 0 differ in respect to the presence of anti-A antibodies in the latter. It is possible that these anti-A antibodies interact with endogenous vWF, thus affecting the half-life time of the factor VIII/vWF complex.

Thrombotic thrombocytopenic purpura (TTP) after bone marrow transplantation (BMT) differs from classic TTP in its clinical course and therapy. A characteristic of classic TTP is the inhibition of a plasma protease that specifically cleaves von Willebrand factor (vWF), thus reducing its multimeric size. We investigated whether this protease was also inhibited in BMT-associated TTP. Plasma from patients with classic or BMT-associated TTP was incubated with recombinant vWF R834Q, a vWF mutant with enhanced sensitivity to the protease. The proteolysis of vWF multimers was analyzed and quantified on Western blot. Metalloprotease activity was strongly inhibited in the classic TTP patient group. However, metalloprotease activity was normal in the BMT-associated TTP patient group. The difference in activity between the two patient groups was highly significant (P = .0016). The results indicate that the etiologies of classic and BMT-associated TTP are indeed different and provide an explanation for the lack of success of plasma exchange in BMT-associated TTP.

Lining the luminal surface of prosthetic vascular grafts with endothelial cells (cell seeding) will lower its thrombogenicity. Commonly used macrovascular human adult endothelial cells (HAEC) require in vitro cultivation before large enough numbers are obtained to cover grafts confluently. Fat-derived microvascular endothelial cells (MVEC) prove to be a good alternative as they can be harvested in much larger numbers while showing similar antithrombotic and fibrinolytic characteristics. An important anticoagulant function of macrovascular endothelial cells is due to the activity of thrombomodulin (TM) on their surface. In this study, the presence and functional activity of TM on fat-derived microvascular cells used in cell seeding was investigated. The expression and localization of TM on MVEC was studied using immunohistochemistry. Functional activity of TM on MVEC was measured by the generation of activated protein C (APC) and was compared to human umbilical vein endothelial cells (HUVEC). TM activity was studied in MVEC seeded on expanded polytetrafluorethylene (ePTFE) vascular prostheses and compared to blank prostheses. We found that TM was expressed on the surface of MVEC, both in vivo and vitro. TM-dependent generation of APC differed significantly between MVEC and HUVEC (3.98 ± 1.2 vs. 3.0 ± 0.7 nM, respectively). After seeding MVEC on vascular prostheses, TM activity did not change. APC generation was significantly higher on MVEC-seeded vascular grafts compared to blank grafts (4.0 ± 0.7 vs. 1.7 ± 0.5 nM, respectively). We conclude that TM is present and highly active on cultured MVEC. When seeded on ePTFE, MVEC retain the possibility to inhibit thrombin coagulant activity and to activate protein C. Therefore, since MVEC are readily available, the anticoagulant properties demonstrated here indicate that this cell type is suitable for cell seeding of vascular prostheses.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

SummaryType 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (vWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 μg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis.

Objectives: To investigate the influence of mesothelial cell (MC) seeding on patency and neointimal formation of small diameter ePTFE grafts in a canine model. Materials and Methods: MC were isolated from the omentum, cultured, seeded on fibronectin-coated ePTFE grafts (4 cm, 4 mm ID), and implanted in the carotid artery of five Beagle dogs. Each dog also received a non-seeded control graft. Patency was assessed by palpation immediately after implantation, and non-invasively by magnetic resonance angiography (MRA) after 1 week and just prior to sacrifice (4 weeks). Intimal thickness was quantified on histological sections by use of computer-aided morphometry. Results: All grafts were patent after implantation. After 1 week, MRA showed the loss of lumen diameter in two seeded grafts. After 4 weeks, two seeded grafts were occluded, one seeded graft was severely stenosed, and all others were without angiographic lumen reduction. Histology and morphometry confirmed that two seeded grafts were occluded, and demonstrated that the other three seeded grafts showed significantly more intima formation (0.22–1.34 mm) than the control grafts ( p Conclusions: The MC seeding process decreases patency and increases neointimal formation of small diameter ePTFE grafts in dogs and does not seem to be useful for reduction of graft thrombogenicity.

SummaryP-selectin is a 140 kD protein found in the α-granules of platelets and the Weibel-Palade bodies of endothelial cells. On cell activation it is expressed on the cell surface and also secreted into plasma. Whether the circulating soluble P-selectin (sP-selectin) originates from platelets, endothelial cells, or both, is not known. We studied the level of sP-selectin in diseases with different platelet counts, with or without evidence of endothelial cell activation. Endothelial cell activation was confirmed by the detection of sE-selectin and EDl-fibronectin. A significant positive correlation between platelet count and sP-selectin concentration was observed in healthy controls, and in patients with thrombocytopenia due to bone marrow aplasia, or with thrombocytosis (r = 0.85; n = 47; p This study suggests that platelets are the major source of circulating sP-selectin in healthy individuals. Endothelial cell activation is associated with an increased sP-selectin concentration per platelet.

Collagen type IV is a sheet-forming collagen and a major constituent of the vessel wall. To find out which conditions are important for platelet adhesion to collagen type IV, we performed perfusion studies with anticoagulated blood in parallel plate perfusion chambers. The role of divalent cations was investigated by using plasmas with variable concentrations of Mg2+ and Ca2+ions. When Mg2+ concentration was decreased from 2.00 mmol/L to 0.25 mmol/L at a fixed Ca2+ concentration of 1.25 mmol/L, platelet coverage on the collagen type IV surface decreased from 22.8% +/-1.8% (n = 4) to 4.6% +/- 0.6% (n = 4) at a shear rate of 1,600 s-1. Also, platelet aggregate formation on collagen type IV was strongly impaired. A monoclonal antibody against the glycoprotein (Gp) Ib receptor and von Willebrand factor (vWF)-depleted plasma reduced the platelet coverage to collagen type IV to, respectively, 10% and 45% of the control value. Electron microscopy showed that vWF was only present between platelets and between the platelet and the collagen type IV surface, but did not bind elsewhere to collagen type IV. These data indicate that collagen type IV is a reactive collagen for platelets. Differences in physiologic plasma magnesium concentrations may in part explain the differences in platelet reactivity to collagen type IV between individuals, and perhaps contribute to differences in the risk for thrombosis.

Co-localization of blood platelets and granulocytes at sites of hemostasis and inflammation has triggered an intense interest in possible interactions between these cellular processes and induction of vessel wall injury. Leukocyte adhesion to endothelial cells decreases with increasing shear and is dependent on an initial rolling phase mediated by selectins. We hypothesized that flow-dependent platelet adhesion at an injured vessel wall will lead to P-selectin expression by platelets, thus mediating leukocyte co-localization. A perfusion chamber was used in which flowing whole blood induced platelet adhesion to a subendothelial matrix (ECM) of cultured human umbilical vein endothelial cells (HUVEC). We compared neutrophil (polymorphonuclear leukocyte [PMN]) interactions with HUVEC and their ECM with and without adhered platelets. PMNs adhered predominantly to ECM-adhered platelets and not to endothelial cells. ECM alone did not support PMN adhesion under flow conditions. PMN adhesion to unstimulated HUVEC was only substantial at low shear (up to 200 cells/mm2 at shear stress 80 mPa). In marked contrast, PMN adhesion to ECM-adhered platelets was dramatically increased, and adhesion was demonstrated at much higher shear stress (up to 640 mPa). Studies with specific antibodies showed that the platelet-dependent neutrophil adhesion was selectin-mediated. Inhibition of P-selectin caused a marked inhibition of adhesion at high shear stress, whereas the role of leukocyte L-selectin was less pronounced. beta2-Integrin-blocking antibodies inhibited static neutrophil adhesion. fMLP induced L-selectin shedding from leukocytes, resulting in decreased leukocyte adhesion. In conclusion, platelet- dependent hemostasis at the ECM appears to be a powerful intermediate in neutrophil-vessel wall interactions at shear stresses that normally do not allow neutrophil adhesion to intact endothelium.

The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N- terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF- dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D′ domain were found to be involved in the protection of factor VIII from inactivation by aPC.

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.

Thermal injury has been shown to reduce platelet adhesion (PA) in vitro but not in vivo. The controversy may be based on the mode of thermal injury, the anticoagulation regimen, or species differences. Human and rabbit arteries were dilated by a radio-frequency (RF)-heated balloon (RF dilation) or by immersion in heated buffer. The artery segments were perfused in an annular perfusion chamber with blood anticoagulant by citrate or heparin (37 degrees C, 5 min, shear rate: 1,300 s-1). To determine PA to deep wall layers, 6-micron cross-sections of heated arteries were perfused in a rectangular perfusion chamber (37 degrees C, 5 min, 1,300 s-1). After RF dilation of human arteries at 55 and 90 degrees C, subendothelical PA (citrated blood) decreased from 28.9% at 37 degrees C to 6.8%, and increased to 39.6%, respectively (in both cases p0.05). Heparin anticoagulation resulted in subendothelial fibrin deposition that was equal after 37 and 90 degrees C, and decreased after 55 degrees C. Heating of cross-sections of atherosclerotic coronary arteries to 55 and 90 degrees C, showed increased and decreased PA, respectively, to the intima and media. No effect was observed on the highly reactive adventitia and atherosclerotic plaque. We conclude that thermal balloon angioplasty at 90 degrees C reduces PA to the arterial subendothelium, but not to the adventitia or the atherosclerotic plaque. As thermal balloon angioplasty in patients will always produce a region with increased PA at 55 degrees C and as heparin anticoagulation permits fibrin deposition that is not affected by heat, it is unlikely that thermal balloon angioplasty alone will reduce thrombotic complications.

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.

Abstract Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP). This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions. In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions. rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC 50 values of 70, 600, and 90 nmol/L, respectively (shear rate=1600 s −1 ). Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s −1 . rLAPP caused a concentration-dependent inhibition of binding of 125 I–von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours. Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect. Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 μmol/L rLAPP). rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells. rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.

Platelet adhesion to purified surface-immobilized fibronectin under flow conditions was investigated. Fibronectin was found to support attachment and spreading of platelets. The extent of platelet spreading depended on the amount of immobilized fibronectin. An antiglycoprotein (anti-GP) IIb/IIIa antibody and an Arg-Gly-Asp (RGD)-containing peptide inhibited adhesion almost completely, whereas antibodies directed against platelet GP Ic/IIa (very late antigen 5) inhibited by 50%. Similar results with the antibodies and the peptide were found in a static system. A comparison of different anticoagulants showed no difference in adhesion using citrate or hirudin. However, unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) as the only anticoagulant or in combination with citrate maximally inhibited adhesion by 80% and 60%, respectively. Preincubation of the immobilized fibronectin with UFH resulted in a maximal inhibition of 90%, whereas preincubation with LMWH had no effect. When we preincubated the surface with heparins of different size, we observed 40% inhibition of adhesion with heparins with an average MW of up to 18 kD, whereas a heparin with an average MW of 21 kD almost completely blocked adhesion. These results indicate that platelet adhesion to fibronectin in flow involves several receptors, is highly RGD-mediated, does not require physiologic levels of divalent cations, and can be inhibited by direct binding of heparin to the fibronectin surface.

An 82-year-old man with a low-grade malignant non-Hodgkin lymphoma and an IgG3 lambda monoclonal gammopathy presented a recently acquired bleeding tendency, characterized by recurrent epistaxis, easy bruising, and episodes of melena, requiring packed red blood cell transfusions. Coagulation studies showed a von Willebrand factor (vWF) defect (Ivy bleeding time, > 15 minutes; vWF antigen [vWF:Ag], 0.08 U/mL; ristocetin cofactor activity [vWF:RCoF], < 0.05 U/mL; collagen binding activity [vWF:CBA], 0.01 U/mL; absence of the high molecular weight multimers of vWF on multimeric analysis). Mixing experiments suggested the presence of an inhibitor directed against the vWF:CBA activity of vWF without significantly inhibiting the FVIII:C, vWF:Ag, and vWF:RCoF activities. The inhibitor was identified as an antibody of the IgM class by immunoabsorption of vWF and inhibitor-vWF complexes from the plasma of the patient. Subsequent immunoprecipitation experiments using recombinant fragments of vWF showed that the inhibitor reacted with both the glycoprotein Ib binding domain (amino acids [aa] 422–826) and the A3 (aa 909–1112) domain of vWF, but not with the A2 (aa 716–908) or D4 (aa 1183–1535) domains. We conclude that the IgM autoantibody inhibits the vWF:CBA activity by reacting with an epitope present on both the glycoprotein Ib and A3 domains of vWF.

An 82-year-old man with a low-grade malignant non-Hodgkin lymphoma and an IgG3 lambda monoclonal gammopathy presented a recently acquired bleeding tendency, characterized by recurrent epistaxis, easy bruising, and episodes of melena, requiring packed red blood cell transfusions. Coagulation studies showed a von Willebrand factor (vWF) defect (Ivy bleeding time, > 15 minutes; vWF antigen [vWF:Ag], 0.08 U/mL; ristocetin cofactor activity [vWF:RCoF], < 0.05 U/mL; collagen binding activity [vWF:CBA], 0.01 U/mL; absence of the high molecular weight multimers of vWF on multimeric analysis). Mixing experiments suggested the presence of an inhibitor directed against the vWF:CBA activity of vWF without significantly inhibiting the FVIII:C, vWF:Ag, and vWF:RCoF activities. The inhibitor was identified as an antibody of the IgM class by immunoabsorption of vWF and inhibitor-vWF complexes from the plasma of the patient. Subsequent immunoprecipitation experiments using recombinant fragments of vWF showed that the inhibitor reacted with both the glycoprotein Ib binding domain (amino acids [aa] 422–826) and the A3 (aa 909–1112) domain of vWF, but not with the A2 (aa 716–908) or D4 (aa 1183–1535) domains. We conclude that the IgM autoantibody inhibits the vWF:CBA activity by reacting with an epitope present on both the glycoprotein Ib and A3 domains of vWF.

Platelet membrane glycoprotein IV (GPIV) is a cell-surface glycoprotein that has been proposed as a receptor for collagen. Recently, it has been shown that platelets with the Naka-negative phenotype lack GPIV on their surface, whereas donors with this phenotype are healthy and do not suffer from hematologic disorders. In this study, we compared Naka- negative platelets with normal platelets in adhesion to collagen types I, III, IV, and V and the extracellular matrix of endothelial cells (ECM) under static and flow conditions. No differences in platelet adhesion and subsequent aggregate formation on the collagens types I, III, and IV were observed under static and flow conditions. Adhesion of both homozygous and heterozygous Naka-negative platelets to collagen type V was strongly reduced under static conditions. Collagen type V was not adhesive under flow conditions. No difference in platelet adhesion to ECM was observed, which suggests that GPIV is not important in adhesion to subendothelium, for which ECM may serve as a model. These results indicate that GPIV is not a functional receptor for collagen under flow conditions.

A human ex vivo thrombosis model was used to investigate whether recombinant annexin V (rANV) can prevent thrombus formation under venous and arterial blood flow conditions. In this model, blood from an antecubital vein of healthy donors was allowed to flow directly over the extracellular matrix of tumor necrosis factor-stimulated endothelial cells (TNF-ECMs). TNF-ECMs were preincubated with rANV (2.9 mumol/L) for 30 minutes. With this rANV concentration all binding sites present on TNF-ECMs (1.6 +/- 0.5 x 10(12)/cm2) are occupied, and a maximal inhibition was observed in a tissue factor-dependent clotting assay. Fibrin deposition and platelet and leukocyte adhesion were measured on the rANV-treated and nontreated TNF-ECMs. Nontreated TNF-ECMs were used as controls. rANV inhibited fibrin deposition by 81% at a wall shear rate of 100 s-1. A nonsignificant inhibition was also observed at 650 s-1. Platelet-matrix adhesion, which is more prominent at higher shear rates, was significantly decreased by 60% at 100 s-1 but not at 650 s-1. The average leukocyte adherence was nonsignificantly lowered at 100 s-1. Virtually no leukocytes adhered at 650 s-1. The results demonstrated that rANV can inhibit blood coagulation under venous blood flow conditions and may serve as an antithrombotic drug.

Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.

A ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-microns cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. The central lipid core of an advanced plaque was not reactive towards platelets. The results indicate that the atherosclerotic plaque by itself is more thrombogenic than the normal vessel wall. To study the cause of the increased thrombus formation on the atherosclerotic plaque, perfusion studies were combined with immunohistochemical studies. Immunohistochemical studies of adhesive proteins showed enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. Laminin and collagen type IV were not enriched. von Willebrand Factor (vWF) was not present in the plaque. The pattern of increased platelet deposition in serial cross sections corresponded best with areas in which collagen types I and III were enriched, but there were also areas in the plaque where both collagens were enriched but no increased reactivity was seen. Inhibition of platelet adhesion with a large range of antibodies or specific inhibitors showed that vWF from plasma and collagen types I and/or III in the plaque were involved. Fibronectin from plasma and fibronectin, fibrinogen, laminin, and thrombospondin in the vessel wall had no effect on platelet adhesion. We conclude that the increased thrombogenicity of atherosclerotic lesions is due to changes in quantity and nature of collagen types I and/or III.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

BACKGROUND Thermal angioplasty methods heat the arterial wall. We related platelet adhesion to the temperature to which subendothelium and purified adhesive proteins had been exposed. METHODS AND RESULTS Cultured subendothelium, purified von Willebrand factor, collagen types I and III, or fibronectin was applied to glass coverslips. Coverslips were mounted on a heating device that applied a temperature gradient from 30 to 100 degrees C. De-endothelialized umbilical arteries were heated by immersion in phosphate-buffered saline. After cooling to room temperature, the surfaces were perfused with blood at 37 degrees C (shear rate, 1600 sec-1). Compared with 37 degrees C, platelet adhesion to endothelial cell matrix was significantly reduced by 25%, 50% or 75% after heating to 69 +/- 1 degree C (mean +/- SEM, P < .05), 72 +/- 1 degree C, or 75 +/- 1 degree C, respectively. Platelet coverage to umbilical artery subendothelium was in the same way significantly reduced after heating to 71 +/- 1 degree C, or 77 +/- 1 degree C, respectively. In contrast to endothelial cell matrix, however, heating to about 55 degrees C increased platelet coverage from 30 +/- 5% to 54 +/- 6% (P < .05). Both platelet adhesion to von Willebrand factor and monoclonal antibody binding against the GpIb binding site of von Willebrand factor showed a comparable temperature dependence as platelet adhesion to subendothelium, provided the proper von Willebrand factor concentration was used. Platelet adhesion to heated collagen types I and III was increased and maximal at 57 +/- 2 degrees C and 62 +/- 2 degrees C, respectively. Preincubation of collagen III with proteins resulted in decreased platelet adhesion with increasing temperatures. Heating did not affect the reactivity of fibronectin. CONCLUSIONS In vitro platelet adhesion to human subendothelium is reduced by more than 50% after heating it briefly to more than 74 degrees C. Temperatures in excess of 80 degrees C reduce platelet adhesion by at least 85%. Thermal denaturation of von Willebrand factor is responsible not only for the decreased thrombogenicity above 71 degrees C but also for the increased thrombogenicity near 55 degrees C, provided that the von Willebrand factor concentration is low.

The effect of D-Arg-Gly-Asp-Trp (dRGDW), a synthetic RGD-containing peptide, on platelet adhesion and aggregate formation on various purified adhesive proteins and the extracellular matrix of endothelial cells was investigated with anticoagulated blood recirculating through a parallel-plate perfusion chamber. Aggregate formation on the extracellular matrix of phorbol myristate acetate (PMA)-stimulated endothelial cells and on collagen type I was more strongly inhibited by dRGDW at higher shear rates than at a low shear rate. Platelet adhesion to the extracellular matrix of nonactivated and PMA-stimulated endothelial cells was inhibited by dRGDW, especially at high shear rates, probably as a consequence of the inhibition of platelet spreading. Inhibition by dRGDW of platelet adhesion to von Willebrand factor, fibronectin, and fibrinogen was almost complete, indicating that platelet adhesion to these substrates is mediated through RGD-directed receptors. Platelet adhesion to laminin was not inhibited by the peptide, whereas platelet adhesion to collagen was increased as a consequence of the inhibition of aggregate formation. Our results show that dRGDW is a strong inhibitor of platelet adhesion and aggregate formation, especially at high shear rates.

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the α-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15000 to 20000. The CD63 MoAb recognize a 30–60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.

Cell seeding may decrease the thrombogenicity of implanted vascular grafts, but its application is hampered by the limited availability of autologous endothelial cells. We studied the interaction of alternate cells, human peritoneal mesothelial cells, with whole blood in a flow chamber. When citrated blood was perfused over mesothelial cells, platelet adhesion was seen on the intercellular matrix but not on the cells themselves. Perfusions with blood anticoagulated with low-molecular-weight heparin resulted in fibrin formation at the surface of mesothelial cells but not at the surface of human umbilical venous endothelial cells. At shear rates of 200 sec-1 fibrin deposition on the mesothelial cell surface increased during the first 5 minutes to 5.7 +/- 1.06 micrograms fibrin per square centimeter, whereafter these values stabilized. The procoagulant activity of cultured mesothelial cells was higher than that of peritoneal membrane studied ex vivo. However, cultured mesothelial cells incubated with polyclonal antibodies against tissue factor showed a significant decrease in procoagulant activity. We conclude that human peritoneal mesothelial cells may be used for cell seeding procedures, provided that their tissue factor expression can be controlled.

Platelet adhesion to the vessel wall is initiated by transport of blood platelets from the bulk flow to the wall. The process of diffusion and convection of the platelets is affected by rheological conditions such as well shear rate, red blood cell (RBC) deformability, and viscosity of the medium. To study the effect of plasma viscosity on platelet adhesion, perfusion experiments with a rectangular perfusion chamber were performed. Reconstituted blood, consisting of washed platelets and washed RBCs, was circulated through this chamber for 5 minutes at a wall shear rate of 300 s-1. Different albumin concentrations were made, to obtain different medium viscosities (0.89 to 1.85 mPa.s). Platelet adhesion decreased with increasing medium viscosity up to viscosities of 0.95 mPa.s, but increased with medium viscosity above this value. Instead of human albumin solution, different plasma viscosities were obtained by dilution of Waldenstrom plasma with buffer. Plasma was depleted of fibronectin, which gave a final plasma viscosity of 2.0 mPa.s, and was dialyzed against HEPES buffer and subsequently diluted with the dialysis buffer in different fractions (0.89 to 2.00 mPa.s). Perfusions were performed over a purified von Willebrand factor coating on glass, or over an endothelial cell matrix, preincubated with von Willebrand factor. With both surfaces, platelet adhesion was dependent on the plasma viscosity in a similar way: at low plasma viscosities, adhesion was decreased with increasing plasma viscosity, while at higher plasma viscosities, adhesion increased with plasma viscosity. Adhesion values at higher plasma viscosity or at higher human albumin concentrations could be explained by effects of the medium on the rigidity of the RBCs, since platelet adhesion is known to be increased by enhanced RBC rigidity. Effects of the medium on the deformability of the RBCs were measured separately with the laser diffraction method. These experiments confirmed that presence of human albumin or plasma in the measuring suspension increased the rigidity of RBCs. To prevent influence of the medium on the RBCs in perfusion experiments, the RBCs were fixated with glutaraldehyde. Perfusion experiments with fixated RBCs in plasma over a von Willebrand factor preincubated endothelial cell matrix, showed a consequent decrease in adhesion with increasing plasma viscosity, according to the diffusion theories, whereas the increase of adhesion at high plasma viscosities was lacking. This suggests that the latter effect was entirely due to increased transport of platelets by more rigid RBCs.

The adhesion of platelets to purified laminin under flow conditions was investigated. Adhesion to laminin was strongly dependent on the presence of divalent cations. In the absence of cations platelet adhesion (8% coverage in 5 minutes) was maximal at a shear rate of 100/s and no adhesion could be detected at shear rates above 800/s. In the presence of 0.8 mmol/L Mg2+ and 2 mmol/L Ca2+ platelet adhesion reached its maximum (30% coverage) around 800/s. At 1,800/s platelets still adhered to purified laminin (coverage of 6%). Antibodies against the E8 domain of laminin and antibodies against the alpha 6 and beta 1 chains of platelet membrane glycoprotein very late activation antigen-6 (VLA-6), completely inhibited adhesion. No inhibition was found with antibodies against glycoprotein IIb:IIIa, against the alpha 2 chain of VLA-2, and against the alpha 5 chain of VLA-5. Fibronectin and von Willebrand factor were not involved in laminin-dependent adhesion. Anti- VLA-6 partly inhibited platelet adhesion to the extracellular matrix of endothelial cells at shear rates below 800/s. Preincubation of the matrices with antilaminin E8 antibodies did not influence the adhesion. These results show that purified laminin supports platelet adhesion and that the presence of VLA-6 is important for platelet adhesion under flow conditions. The protein in the matrix with which VLA-6 interacts is currently unknown.

The adhesion of platelets to purified laminin under flow conditions was investigated. Adhesion to laminin was strongly dependent on the presence of divalent cations. In the absence of cations platelet adhesion (8% coverage in 5 minutes) was maximal at a shear rate of 100/s and no adhesion could be detected at shear rates above 800/s. In the presence of 0.8 mmol/L Mg2+ and 2 mmol/L Ca2+ platelet adhesion reached its maximum (30% coverage) around 800/s. At 1,800/s platelets still adhered to purified laminin (coverage of 6%). Antibodies against the E8 domain of laminin and antibodies against the alpha 6 and beta 1 chains of platelet membrane glycoprotein very late activation antigen-6 (VLA-6), completely inhibited adhesion. No inhibition was found with antibodies against glycoprotein IIb:IIIa, against the alpha 2 chain of VLA-2, and against the alpha 5 chain of VLA-5. Fibronectin and von Willebrand factor were not involved in laminin-dependent adhesion. Anti- VLA-6 partly inhibited platelet adhesion to the extracellular matrix of endothelial cells at shear rates below 800/s. Preincubation of the matrices with antilaminin E8 antibodies did not influence the adhesion. These results show that purified laminin supports platelet adhesion and that the presence of VLA-6 is important for platelet adhesion under flow conditions. The protein in the matrix with which VLA-6 interacts is currently unknown.

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

SummaryThe effect of sera and IgG from 12 patients with systemic lupus erythematosus (SLE) on the endothelial cell (EC) procoagulant activity (PCA) was investigated in an in vitro thrombosis model. Six of the 12 SLE sera contained antiphospholipid antibodies (aPL).EC were stimulated for 8 h at 37° C with or without 50 pM tumor necrosis factor (TNF) in culture medium containing 20% patient or control serum. Then the endothelial cell matrix (ECM) was isolated and subsequently exposed in a perfusion chamber to circulating normal whole blood, anticoagulated with low molecular weight heparin (LMWH). The PCA of the ECM was determined as the amount of generated fibrinopeptide A (FPA) in samples taken before and after perfusion. Furthermore, cross sections were made of the perfused matrix and analyzed for platelet adhesion and aggregate formation.All six aPL containing sera induced a small, but significant increase of ECM procoagulant activity. When added in combination with a low dose of TNF (50 pM), a synergistic enhancement of ECM procoagulant activity was found. The FPA generation was increased to 150–614% from the values obtained after stimulation with TNF and control serum. Also a shift towards the formation of larger platelet thrombi was observed. After stimulation with TNF and patient serum the surface of ECM covered with large aggregates (> 5 µm) was increased by 124–329% compared to the results obtained after stimulation with control serum and TNF. When patient sera were depleted from IgG the effects were strongly decreased.These data show that the potentiation of TNF-induced PCA formation by aPL containing sera from patients with SLE leads to enhanced thrombus formation in an in vitro thrombosis model. This may help explain the increased thrombotic tendency in these patients.

In a prospective double-blind trial, we treated 194 patients with acute venous thromboembolism with heparin or low molecular weight heparin (LMWH; Fragmin). To evaluate the most important prognostic factors for bleeding, the presenting clinical features of the patients, the patients' anticoagulant responses, and the doses of the drugs were analyzed using univariate and multivariate regression analyses. No significant differences in clinical risk factors associated with bleeding were observed between heparin and LMWH. The univariate analyses ranked the parameters in the following order of importance: World Health Organization (WHO) performance status, history of bleeding tendency, cardiopulmonary resuscitation, recent trauma or surgery, leukocyte counts, platelet counts, duration of symptoms, and body surface area. Patients with WHO grade 4 had an eightfold increase in risk of bleeding as compared with WHO grade 1. Assessment of the individual contribution of each variable using multivariate regression analysis showed that the WHO performance status was the most important independent factor predicting major bleeding. A history of a bleeding tendency, recent trauma or surgery, and body surface area were also independent risk factors. The risk of bleeding was influenced by two factors related to the treatment, the patient's anticoagulant response as measured with the anti-Xa assay and the dose of the drug expressed as U/24 h/m2. An increased risk of bleeding was only observed at mean anti-Xa levels greater than 0.8 U/mL for both drugs. Significantly more major bleedings occurred in patients treated with high doses of the drugs, an observation that was independent of the concomitant anti-Xa levels. It should be considered whether choosing an appropriate initial dose adapted to the patient's body surface area and clinical risk factors can improve the efficacy to safety ratio of heparin treatment.

Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.

Uremia is associated with bleeding diathesis. Platelet adhesion to the subendothelium is inhibited by a factor in uremic plasma that may play a role in the disturbed hemostasis of uremic patients. In the formation of the hemostatic plug, platelet adherence is followed by stimulus-induced platelet aggregation and reinforcement by thrombin-generated fibrin. To study these processes in uremic blood, a newly developed thrombosis model was used. Perfusates anticoagulated with low-molecular-weight heparin were circulated over a matrix of stimulated cultured endothelial cells. By stimulation of the endothelial cells, tissue factor was synthesized and deposited in the matrix. When this tissue factor rich-matrix was exposed to flowing blood, local thrombin was formed via activation of the extrinsic coagulation pathway. With this system, platelet adhesion, thrombin-dependent platelet activation, and fibrin formation can all be studied at the same surface. In addition to an adhesion defect, decreased aggregate formation was also found in uremic perfusates. Normal platelets in uremic plasma showed similar results, which indicates that a factor in uremic plasma caused this adhesion and aggregation defect. Platelet aggregation in the system was dependent on endogenously formed thrombin. Fibrinopeptide A generation, however, was normal in uremic perfusates; therefore, uremic plasma has a normal capacity to form thrombin. Resuspension of washed uremic platelets in control plasma did not reverse the aggregation defect in perfusions. In contrast, aggregometer studies with isolated uremic platelets could not detect an abnormal response to threshold concentrations of exogenous thrombin. Thus, uremic toxin(s) cause defective aggregate formation in flow, but not necessarily in the aggregometer. This apparent discrepancy may be due to the higher shear forces in the flow system, which may prevent aggregate formation that is allowed in the aggregometer. Another explanation, that uremic platelets are less responsive to locally formed thrombin than they are to exogenously added thrombin, seems less likely.

To study fibrin incorporation into thrombi at different wall shear rates, a new method to study fibrin deposition on extracellular matrixes underlying stimulated endothelial cells under flow conditions was developed. For this method, we used fibrinogen labeled with peroxidase (Fg-PO). Fg-PO was fully exchangeable for Fg in the clotting assays tested, and PO activity was bound to fibrin-specific fragments. Fg-PO containing fibrin could be stained for microscopic studies with 3,3'-diaminobenzidine and could be quantified by oxidation of phenylenediamine. The absorbance values at 492 nm were converted to fibrin quantities via a standard curve. To study fibrin deposition, Fg-PO was added in trace amounts to whole blood anticoagulated with low-molecular-weight heparin, and perfusion studies were performed over endothelial cell matrixes containing tissue factor. In parallel perfusion studies, 125I-labeled Fg was added in trace amounts to whole blood instead of Fg-PO. Both quantitative methods demonstrated decreased fibrin deposition after perfusions at 1,300 sec-1 compared with fibrin deposition after perfusions at 300 sec-1, while fibrinopeptide A generation was independent of the wall shear rate. The decrease in fibrin deposition at 1,300 sec-1 was accompanied by the appearance of fibrin monomers in the perfusate. This suggested that the decrease in fibrin incorporation at 1,300 sec-1 was due to the impaired polymerization of fibrin monomers. This impairment was probably due to a decrease in local fibrin monomer concentration as a result of the increased removal of monomers from the surface at 1,300 sec-1.

SummaryIn the present study, uremic patients on chronic maintenance hemodialysis were treated with recombinant erythropoietin. Before and after 20 weeks of treatment, platelet adhesion and aggregation were studied with perfusions over a sprayed collagen surface and over matrix of cultured endothelial cells with high tissue factor activity. The influence of the erythropoietin induced raise in hematocrit on platelet transport and adhesion was excluded by performing the perfusions at a standard red blood cell concentration. The present study clearly demonstrates that erythropoietin treatment improves platelet adhesion and aggregation in addition to and independent of its effect on the hematocrit.Studies with control platelets resuspended in plasma of untreated patients showed that a uremic plasma factor reduced adhesion and thrombin- and collagen-dependent aggregation. Patient platelets resuspended in control plasma showed no defects. After erythropoietin treatment, the plasma-induced inhibition of adhesion and aggregation had almost completely disappeared from patient plasma.The beneficial effect of the erythropoietin treatment on uremic hemostasis is therefore twofold. The increase of the red blood cell mass improves transport of platelets, and thus adhesion to the vessel wall. The intrinsic defect due to the presence of an inhibitory toxin in uremic plasma is, in large part, corrected. Improved neutralization of uremic toxins by red blood cells or less production of toxins by better oxygenated tissue might play a role in the observed phenomena.

Balloon angioplasty produces mechanical vessel wall injury that leads to substantial blood platelet deposition at the angioplasty site. The aim of the study was to determine whether thermal angioplasty might, by contrast, reduce platelet adhesion by denaturation of subendothelial adhesive proteins.Native and cultured human subendothelium was briefly heated to greater than or equal to 100 degrees C by laser irradiation or to 50-100 degrees C by immersion in preheated phosphate buffered saline (PBS). Subsequently, the subendothelium was exposed for 5 min to flowing human blood at a shear rate of 1300 s-1. Blood platelet adhesion to the subendothelium was determined quantitatively.Human umbilical arteries were used and the subendothelial matrix derived from cultured umbilical vein endothelial cells.After heating arterial subendothelium by laser irradiation to greater than or equal to 100 degrees C, zero platelet adhesion was found v 36(SD 2)% adhesion to the non-heated surface (p less than 0.001). After laser heating of the subendothelial matrix to greater than or equal to 100 degrees C, platelet adhesion was absent in 10/10 experiments (p less than 0.01). After heating the matrix to 100 degrees C by immersion in PBS, platelet adhesion was reduced to 5(5)% v 31(7)% at 37 degrees C (p less than 0.001).These in vitro results, if extrapolated to catheter interventions, suggest that thermal injury to the vessel wall by laser angioplasty or other thermal angioplasty methods may provide a basic and clinically relevant advantage over mechanical angioplasty modalities, because of a potentially reduced risk of complications related to platelet adhesion.

Platelet deposition on cultured fibroblasts and on their extracellular matrix (FBM) was investigated in a flow system with citrated blood and was compared with platelet deposition on cultured endothelial cells, smooth muscle cells, and their extracellular matrices. Platelet deposition was present at all surfaces except on intact endothelial cells. Deposition on FBM consisted of contact platelets, spread platelets, and a few small aggregates. On intact fibroblasts cells, the surface coverage was lower, and platelets formed aggregates. Factors involved in primary hemostasis, particularly the wall shear rate, von Willebrand factor (vWF), and fibronectin, were investigated on FBM. The reactivity of FBM was determined by the passage number of the cultured cells. The vWF was involved in platelet adhesion on FBM at only the high shear rate (greater than 800 s-1). Platelet deposition was independent of plasma fibronectin at all shear rates tested. Matrix-associated fibronectin was involved in adhesion at low and high wall shear rates. We conclude that FBM can be used as a platelet adhesive surface especially to study the contribution of exogenous vWF to platelet adhesion because FBM does not contain vWF.

When the endothelial cell layer is damaged, a thrombotic reaction starts on the cells' subendothelium and on the connective tissue deposited by smooth muscle cells in the deeper layers. When more severe vascular damage occurs, hemostasis will involve the vessel adventitia in which fibroblasts are found. In this article, the influence of in vitro cultured endothelial cells, smooth muscle cells, and fibroblasts on the hemostatic balance was studied. To do so, perfusions were performed with low molecular weight heparin anticoagulated blood over the extracellular matrix of the cells. This method allowed the study of tissue factor-dependent thrombin generation and its influence on formation of fibrin and platelet aggregates. The experiments described in this article show that endothelial cells isolated from different human organs interfere differently in the hemostatic response. Endothelial cells isolated from umbilical veins are nonthrombogenic; they do not synthesize tissue factor under unstimulated conditions. On their extracellular matrix, only adherent platelets are found, but no aggregates and no fibrin. Endothelial cells isolated from omentum and atrium contain tissue factor activity under unstimulated conditions. As a consequence, thrombin is generated on their surfaces, and platelet aggregates and fibrin deposition are found on the extracellular matrices after perfusions with whole blood. The matrix of smooth muscle cells and fibroblasts behaved similarly. Increase in shear rate and perfusion time resulted in an increase in platelet aggregate formation. Polymerized fibrin deposition decreased when perfusions were performed at higher shear. Both platelet aggregation and fibrin deposition were tissue factor dependent and could be blocked more than 70% by an antibody against tissue factor. Based on these results, we conclude that endothelial cells isolated from umbilical veins form the best nonthrombogenic surface in vitro. Moreover, coagulation-dependent hemostasis should be included when thrombogenicity of subendothelium is discussed, especially when it concerns matrix derived from cells present in the deeper layer of the vessel wall.